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The N-Glycan Cluster from Xanthomonas campestris pv. campestris

A TOOLBOX FOR SEQUENTIAL PLANT N-GLYCAN PROCESSING*
  • Stéphanie Dupoiron
    Footnotes
    Affiliations
    Université de Toulouse, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France
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  • Claudine Zischek
    Footnotes
    Affiliations
    INRA, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire des Interactions Plantes-Microorganismes, UMR 2594, F-31326 Castanet-Tolosan, France
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  • Laetitia Ligat
    Footnotes
    Affiliations
    Université de Toulouse, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France
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  • Julien Carbonne
    Affiliations
    Université de Toulouse, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France
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  • Alice Boulanger
    Footnotes
    Affiliations
    INRA, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire des Interactions Plantes-Microorganismes, UMR 2594, F-31326 Castanet-Tolosan, France
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  • Thomas Dugé de Bernonville
    Footnotes
    Affiliations
    INRA, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire des Interactions Plantes-Microorganismes, UMR 2594, F-31326 Castanet-Tolosan, France
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  • Martine Lautier
    Affiliations
    INRA, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire des Interactions Plantes-Microorganismes, UMR 2594, F-31326 Castanet-Tolosan, France

    Université de Toulouse, UPS, F-31062 Toulouse, France
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  • Pauline Rival
    Footnotes
    Affiliations
    Université de Toulouse, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    INRA, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire des Interactions Plantes-Microorganismes, UMR 2594, F-31326 Castanet-Tolosan, France
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  • Matthieu Arlat
    Affiliations
    INRA, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire des Interactions Plantes-Microorganismes, UMR 2594, F-31326 Castanet-Tolosan, France

    Université de Toulouse, UPS, F-31062 Toulouse, France
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  • Elisabeth Jamet
    Affiliations
    Université de Toulouse, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France
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  • Emmanuelle Lauber
    Correspondence
    To whom correspondence should be addressed: Laboratoire des Interactions Plantes-Microorganismes, UMR 2594/441, Chemin de Borde Rouge, CS52627, F-31326 Castanet-Tolosan, France, Tel.: 33-5-6128-5047; Fax: 33-5-6128-5061
    Affiliations
    INRA, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire des Interactions Plantes-Microorganismes, UMR 2594, F-31326 Castanet-Tolosan, France
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  • Cécile Albenne
    Affiliations
    Université de Toulouse, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France

    CNRS, Laboratoire de Recherches en Sciences Végétales, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France
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  • Author Footnotes
    * This work was supported by French Agence Nationale de la Recherche Grant ANR-08-BLAN-0193-01, the local Fédération de Recherche Agrobiosciences, Interactions et Biodiversité, the Université Paul Sabatier, the Centre National de la Recherche Scientifique, the Institut National de la Recherche Agronomique, and the French Ministry of Research and Technology (to A.B.).
    1 Both authors contributed equally to this work.
    2 Present address: AgroParisTech, Chaire Agro-Biotechnologies Industrielles, 247 Rue Paul Vaillant Couturier, F-51100 Reims, France.
    3 Present address: INSERM UMR1037, Plateforme Protéomique, Pôle Technologique du Centre de Recherches en Cancérologie de Toulouse, 2 Avenue Hubert Curien, F-31100 Toulouse, France.
    4 Present address: Laboratoire de Chimie Bactérienne, CNRS UMR 7283, F-13009 Marseille, France.
    5 Present address: EA2106, Biomolécules et Biotechnologies Végétales, UFR Sciences et Techniques, Université François Rabelais de Tours, 37000 Tours, France.
    6 Present address: Dept. of Genome Sciences, University of Washington, Seattle, WA 98195.
Open AccessPublished:January 13, 2015DOI:https://doi.org/10.1074/jbc.M114.624593
      N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N2M3FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the β-xylosidase NixI (GH3), which is involved in the cleavage of the β-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.

      Introduction

      In the context of host-bacteria interactions, i.e. symbiosis or pathogenesis, the question of the degradation of host N-glycoproteins by microorganisms is relevant, and only a few studies demonstrating orchestrated processes involving catabolic enzymes have been reported. Glycosylation is the most widespread post-translational modification of proteins found in nature (
      • Spiro R.G.
      Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.
      ). It concerns not only eukaryotes but also some bacteria and archaea for which several examples of sugar attachment on proteins have been reported (
      • Messner P.
      • Allmaier G.
      • Schäffer C.
      • Wugeditsch T.
      • Lortal S.
      • König H.
      • Niemetz R.
      • Dorner M.
      Biochemistry of S-layers.
      ,
      • Jarrell K.F.
      • Ding Y.
      • Meyer B.H.
      • Albers S.V.
      • Kaminski L.
      • Eichler J.
      N-Linked glycosylation in archaea: a structural, functional, and genetic analysis.
      ). In eukaryotes, two types of glycosylation are described, N- and O-glycosylation. Whereas O-glycosylation can display a huge diversity, in terms of structure and of the sequence surrounding the amino acid carrier, N-glycosylation is more conserved (
      • Faye L.
      • Boulaflous A.
      • Benchabane M.
      • Gomord V.
      • Michaud D.
      Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming.
      ). N-Glycosylation occurs in the secretory pathway, by adding a N-glycan to the amide group of asparagine in the Asn-Xaa-Ser/Thr context, where Xaa can be any amino acid except Pro. It starts in the endoplasmic reticulum by the co-translational transfer of a preformed lipid-linked oligosaccharide onto the nascent polypeptide. Then maturation steps involving glycosidases and glycosyltransferases take place in the Golgi apparatus through secretion of proteins to their final destination. Resulting N-glycans can be of three types, either high mannose-, hybrid-, or complex-type (
      • Faye L.
      • Boulaflous A.
      • Benchabane M.
      • Gomord V.
      • Michaud D.
      Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming.
      ). This global mechanism is common to most eukaryotic systems, but the action of specific maturation enzymes leads to different complex N-glycan structures. For instance, in plants, the first GlcNAc of the core can be substituted with an α-1,3-fucose, instead of an α-1,6-fucose for human N-glycans. In addition, the β-mannose of the core can be decorated with a β-1,2-xylose in plants, whereas a β-1,4-GlcNAc can be found at this position in human glycans. From a functional point of view, N-glycans can modify the folding, the activity, or the stability of proteins and subsequently play key roles in a number of physiological processes, such as signal transduction, targeting, cell-cell recognition, infection, and immunity (
      • Mitra N.
      • Sinha S.
      • Ramya T.N.
      • Surolia A.
      N-Linked oligosaccharides as outfitters for glycoprotein folding, form and function.
      ,
      • Rudd P.M.
      • Elliott T.
      • Cresswell P.
      • Wilson I.A.
      • Dwek R.A.
      Glycosylation and the immune system.
      ,
      • An H.J.
      • Lebrilla C.B.
      A glycomics approach to the discovery of potential cancer biomarkers.
      ).
      Investigation into the alteration of N-glycan structure, because of either aberrant N-glycosylation build-up or degradation, has focused on a better understanding of the molecular basis of these biological processes. N-Glycan degradation involves carbohydrate-active enzymes, in particular glycoside hydrolases (GHs)
      The abbreviations used are: GH
      glycoside hydrolase
      Xcc
      Xanthomonas campestris pv. campestris
      MME
      minimal medium for hrp gene expression
      MBP
      maltose-binding protein
      pNP
      para-nitrophenyl
      pNP-α-Fuc
      pNP-α-l-fucopyranoside
      pNP-β-GlcNAc
      pNP-N-acetyl-β-d-glucosaminide
      pNP-β-GalNAc
      pNP-N-acetyl-β-d-galactosaminide
      pNP-β-Man
      pNP-β-d-mannopyranoside
      pNP-β-Xyl
      pNP-β-d-xylopyranoside
      pNP-α-Man
      pNP-α-d-mannopyranoside
      pNP-β-Gal
      pNP-β-d-galactopyranoside
      CE
      crude extract
      ConA
      concanavalin A.
      presently classified into 133 families (
      • Cantarel B.L.
      • Coutinho P.M.
      • Rancurel C.
      • Bernard T.
      • Lombard V.
      • Henrissat B.
      The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics.
      ). In addition, synergic processes may also occur, in particular involving glycoside phosphorylases (
      • Ladevèze S.
      • Tarquis L.
      • Cecchini D.A.
      • Bercovici J.
      • André I.
      • Topham C.M.
      • Morel S.
      • Laville E.
      • Monsan P.
      • Lombard V.
      • Henrissat B.
      • Potocki-Véronèse G.
      Role of glycoside phosphorylases in mannose foraging by human gut bacteria.
      ,
      • Nihira T.
      • Suzuki E.
      • Kitaoka M.
      • Nishimoto M.
      • Ohtsubo K.
      • Nakai H.
      Discovery of β-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase involved in the metabolism of N-glycans.
      ). To date, the most documented model of glycoconjugate breakdown concerns the gut bacterium Bacteroides thetaiotaomicron, which displays a broad repertoire of endo- and exo-glycosidases designed to hydrolyze plant polysaccharides, as well as human N-glycans (
      • Martens E.C.
      • Koropatkin N.M.
      • Smith T.J.
      • Gordon J.I.
      Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm.
      ,
      • Tailford L.E.
      • Money V.A.
      • Smith N.L.
      • Dumon C.
      • Davies G.J.
      • Gilbert H.J.
      Mannose foraging by Bacteroides thetaiotaomicron: structure and specificity of the β-mannosidase, BtMan2A.
      ,
      • Zhu Y.
      • Suits M.D.
      • Thompson A.J.
      • Chavan S.
      • Dinev Z.
      • Dumon C.
      • Smith N.
      • Moremen K.W.
      • Xiang Y.
      • Siriwardena A.
      • Williams S.J.
      • Gilbert H.J.
      • Davies G.J.
      Mechanistic insights into a Ca2+-dependent family of alpha-mannosidases in a human gut symbiont.
      ,
      • Thompson A.J.
      • Williams R.J.
      • Hakki Z.
      • Alonzi D.S.
      • Wennekes T.
      • Gloster T.M.
      • Songsrirote K.
      • Thomas-Oates J.E.
      • Wrodnigg T.M.
      • Spreitz J.
      • Stütz A.E.
      • Butters T.D.
      • Williams S.J.
      • Davies G.J.
      Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase.
      ). The biological role of GHs involved in N-glycan degradation is unclear, but it may consist of mannose foraging for basic metabolic inputs. In Bacteroides fragilis, the don locus, which confers a selective advantage during extraintestinal infections, is involved in transferrin deglycosylation (
      • Cao Y.
      • Rocha E.R.
      • Smith C.J.
      Efficient utilization of complex N-linked glycans is a selective advantage for Bacteroides fragilis in extraintestinal infections.
      ). Enterococcus faecalis, a nosocomial human pathogen, produces an endo-β-N-acetylglucosaminidase able to release high mannose-type N-glycans from glycoproteins. This activity, associated with mannosidase action, may play a role in the survival and persistence of the pathogen in vivo (
      • Roberts G.
      • Tarelli E.
      • Homer K.A.
      • Philpott-Howard J.
      • Beighton D.
      Production of an endo-β-N-acetylglucosaminidase activity mediates growth of Enterococcus faecalis on a high-mannose-type glycoprotein.
      ). Similarly, Streptococcus oralis, responsible for infection of immunocompromised patients, was shown to deglycosylate complex-type N-glycans through a sequential mechanism. Released monosaccharides are assumed to be used by the bacteria to sustain growth (
      • Byers H.L.
      • Tarelli E.
      • Homer K.A.
      • Beighton D.
      Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human α1-acid glycoprotein mediates growth of Streptococcus oralis.
      ). However, S. oralis enzymes responsible for N-glycan processing have not been identified. More recently, enzymes from Streptococcus pyogenes and Streptococcus pneumoniae classified in the GH38 (α-1,3-mannosidase) and GH125 (α-1,6-mannosidase) families, respectively, have been identified. These enzymes are active on N-glycans, highlighting the processing of N-glycans by Streptococcus bacteria (
      • Suits M.D.
      • Zhu Y.
      • Taylor E.J.
      • Walton J.
      • Zechel D.L.
      • Gilbert H.J.
      • Davies G.J.
      Structure and kinetic investigation of Streptococcus pyogenes family GH38 α-mannosidase.
      ,
      • Gregg K.J.
      • Zandberg W.F.
      • Hehemann J.H.
      • Whitworth G.E.
      • Deng L.
      • Vocadlo D.J.
      • Boraston A.B.
      Analysis of a new family of widely distributed metal-independent alpha-mannosidases provides unique insight into the processing of N-linked glycans.
      ). Finally, it was shown that the human pathogen Capnocytophaga canimorsus deglycosylates surface glycoproteins from the host and supports its growth on the released glycan moiety (
      • Mally M.
      • Shin H.
      • Paroz C.
      • Landmann R.
      • Cornelis G.R.
      Capnocytophaga canimorsus: a human pathogen feeding at the surface of epithelial cells and phagocytes.
      ). A large enzymatic complex has been identified, and a functional model of deglycosylation processing has been proposed (
      • Renzi F.
      • Manfredi P.
      • Mally M.
      • Moes S.
      • Jenö P.
      • Cornelis G.R.
      The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG.
      ).
      Despite fast growing advances in understanding N-glycan processing by human pathogenic bacteria, nothing is known about the capacity of plant pathogens to achieve such mechanisms. In the plant pathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) strain ATCC33913, we have recently identified an operon coding for eight GHs (NixE to NixL; N-acetylglucosamine-induced in Xcc) that belongs to the GlcNAc exploitation system. At the transcriptional level, the expression of nixE to nixL is induced in the presence of GlcNAc, and nixE to nixH are under the control of the LacI family NagR repressor, the repressive effect of which is relieved in the presence of GlcNAc (
      • Boulanger A.
      • Zischek C.
      • Lautier M.
      • Jamet S.
      • Rival P.
      • Carrère S.
      • Arlat M.
      • Lauber E.
      The plant pathogen Xanthomonas campestris pv. campestris exploits N-acetylglucosamine during infection.
      ). This operon was shown to be involved in the exploitation of GlcNAc-containing molecules derived from the plant during infection (
      • Boulanger A.
      • Zischek C.
      • Lautier M.
      • Jamet S.
      • Rival P.
      • Carrère S.
      • Arlat M.
      • Lauber E.
      The plant pathogen Xanthomonas campestris pv. campestris exploits N-acetylglucosamine during infection.
      ). Based on their predicted functions, Nix GHs were proposed to be involved in the cleavage of glycosidic bonds found in plant N-glycans, and the nixE-nixL cluster was called N-glycan cluster (
      • Boulanger A.
      • Zischek C.
      • Lautier M.
      • Jamet S.
      • Rival P.
      • Carrère S.
      • Arlat M.
      • Lauber E.
      The plant pathogen Xanthomonas campestris pv. campestris exploits N-acetylglucosamine during infection.
      ).
      The objective of the present work was to experimentally demonstrate the involvement of Nix enzymes in plant N-glycan degradation. The predicted activities were first confirmed on pNP synthetic substrates, and the kinetic parameters were determined. Then Nix enzymes were shown to sequentially process an N-glycopeptide. Our data provide the first evidence of N-glycan degradation by a plant pathogenic bacterium in vitro and broaden the source of substrates putatively exploited by phytopathogens like Xanthomonas strains during infection. This study constitutes a new example of N-glycan processing by bacteria and provides new insights into the deglycosylation mechanism carried out by GHs.

      DISCUSSION

      Xcc, the causal agent of black rot disease on Brassica plants, produces an extensive repertoire of GHs, lyases, and esterases (
      • da Silva A.C.
      • Ferro J.A.
      • Reinach F.C.
      • Farah C.S.
      • Furlan L.R.
      • Quaggio R.B.
      • Monteiro-Vitorello C.B.
      • Van Sluys M.A.
      • Almeida N.F.
      • Alves L.M.
      • do Amaral A.M.
      • Bertolini M.C.
      • Camargo L.E.
      • Camarotte G.
      • Cannavan F.
      • Cardozo J.
      • Chambergo F.
      • Ciapina L.P.
      • Cicarelli R.M.
      • Coutinho L.L.
      • Cursino-Santos J.R.
      • El-Dorry H.
      • Faria J.B.
      • Ferreira A.J.
      • Ferreira R.C.
      • Ferro M.I.
      • Formighieri E.F.
      • Franco M.C.
      • Greggio C.C.
      • Gruber A.
      • Katsuyama A.M.
      • Kishi L.T.
      • Leite R.P.
      • Lemos E.G.
      • Lemos M.V.
      • Locali E.C.
      • Machado M.A.
      • Madeira A.M.
      • Martinez-Rossi N.M.
      • Martins E.C.
      • Meidanis J.
      • Menck C.F.
      • Miyaki C.Y.
      • Moon D.H.
      • Moreira L.M.
      • Novo M.T.
      • Okura V.K.
      • Oliveira M.C.
      • Oliveira V.R.
      • Pereira H.A.
      • Rossi A.
      • Sena J.A.
      • Silva C.
      • de Souza R.F.
      • Spinola L.A.
      • Takita M.A.
      • Tamura R.E.
      • Teixeira E.C.
      • Tezza R.I.
      • Trindade dos Santos M.
      • Truffi D.
      • Tsai S.M.
      • White F.F.
      • Setubal J.C.
      • Kitajima J.P.
      Comparison of the genomes of two Xanthomonas pathogens with differing host specificities.
      ) that target linkages present in plant cell wall polysaccharides such as cellulose, mannan, xylan, galacturonan, and pectin. In this paper, we characterized Nix GHs belonging to the Xcc GlcNAc utilization system on pNP synthetic substrates, and we demonstrated their ability to degrade a plant N-glycan. This is the first evidence of in vitro N-glycan degradation by a plant pathogen suggesting that N-glycans may by metabolized by phytopathogenic bacteria during infection. By using Xcc total extracts or His-tagged proteins purified from Xcc, we demonstrated that N-glycan processing is sequential. On the N2M3FX N-glycopeptide substrate, NixK initiates the deglycosylation by removing the α-1,3-mannose, NixI acts on the β-1,2-xylose in a second step, and then NixJ releases the α-1,6-mannose. NixE and NixH are assumed to act at a later stage, by removing the α-1,3-fucose and the β-1,4-mannose, respectively. This sequential mode of action corroborates literature data reporting that, in the context of fruit ripening, the removal of α-1,3-mannose from N-glycan is a prerequisite for hydrolysis of the β-1,2-xylose by a tomato xylosidase (
      • Yokouchi D.
      • Ono N.
      • Nakamura K.
      • Maeda M.
      • Kimura Y.
      Purification and characterization of β-xylosidase that is active for plant complex type N-glycans from tomato (Solanum lycopersicum): removal of core alpha1–3 mannosyl residue is prerequisite for hydrolysis of β1–2 xylosyl residue.
      ). In addition, in the human N-glycan degrading Firmicutes, a sequential model is proposed, with the removal of the α-1,3-mannose by a GH38 as a first step necessary for the activity of a GH125 subsequently involved in the removal of the α-1,6-mannose (
      • Gregg K.J.
      • Zandberg W.F.
      • Hehemann J.H.
      • Whitworth G.E.
      • Deng L.
      • Vocadlo D.J.
      • Boraston A.B.
      Analysis of a new family of widely distributed metal-independent alpha-mannosidases provides unique insight into the processing of N-linked glycans.
      ). Such common features suggest that N-glycan processing is of widespread importance, in particular for pathogens in a variety of host contexts.
      In Xcc, although the N-glycan cluster potentially encodes all the enzymes required for complete degradation of the N2M3FX N-glycan, the N2MF product accumulates, suggesting that in our conditions, there is a limiting step for the N2MF glycopeptide degradation process. The reasons for this limiting step may be multiple. Indeed, it could be due to inappropriate condition assays or to the fact that the polypeptidic part of the glycopeptide provides a steric hindrance for NixE, NixF, and/or NixH activities. It is also possible that NixE, NixF, and/or NixH are not in a form active on N-glycans or that NixF is not able to accommodate in its active site the N-glycopeptide used in this study. Furthermore, we cannot exclude that other secreted critical enzyme(s), not encoded by the N-glycan cluster, are required for full N-glycan processing. Finally, this limiting step could be a strategy for the bacterium to partially deglycosylate plant N-glycoproteins. Indeed, in S. pneumoniae strain D39, although the genome codes for α- and β-mannosidases (belonging to GH2, GH38, GH92, and GH125 families; Carbohydrate-Active EnZymes database), deglycosylation of human complex-type N-glycoconjugates is partial with only removal of terminal sialic acid, galactose and GlcNAc residues. This led to the exposure of mannose residues on host glycoproteins, which plays a role in adherence of unencapsulated pneumococci on human cells (
      • King S.J.
      • Hippe K.R.
      • Weiser J.N.
      Deglycosylation of human glycoconjugates by the sequential activities of exoglycosidases expressed by Streptococcus pneumoniae.
      ).
      The results obtained here suggest that in Xcc, as observed in S. oralis (
      • Byers H.L.
      • Tarelli E.
      • Homer K.A.
      • Beighton D.
      Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human α1-acid glycoprotein mediates growth of Streptococcus oralis.
      ) and S. pneumoniae (
      • King S.J.
      • Hippe K.R.
      • Weiser J.N.
      Deglycosylation of human glycoconjugates by the sequential activities of exoglycosidases expressed by Streptococcus pneumoniae.
      ), deglycosylation is sequential because of the action of exo-enzymes. However, in human pathogens such as E. faecalis (
      • Roberts G.
      • Tarelli E.
      • Homer K.A.
      • Philpott-Howard J.
      • Beighton D.
      Production of an endo-β-N-acetylglucosaminidase activity mediates growth of Enterococcus faecalis on a high-mannose-type glycoprotein.
      ), S. pyogenes (
      • Collin M.
      • Olsén A.
      EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG.
      ), B. fragilis (
      • Cao Y.
      • Rocha E.R.
      • Smith C.J.
      Efficient utilization of complex N-linked glycans is a selective advantage for Bacteroides fragilis in extraintestinal infections.
      ), and C. canimorsus (
      • Nihira T.
      • Suzuki E.
      • Kitaoka M.
      • Nishimoto M.
      • Ohtsubo K.
      • Nakai H.
      Discovery of β-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase involved in the metabolism of N-glycans.
      ,
      • Renzi F.
      • Manfredi P.
      • Mally M.
      • Moes S.
      • Jenö P.
      • Cornelis G.R.
      The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG.
      ), and in infant gut-associated Bifidobacteria (
      • Garrido D.
      • Nwosu C.
      • Ruiz-Moyano S.
      • Aldredge D.
      • German J.B.
      • Lebrilla C.B.
      • Mills D.A.
      Endo-β-N-acetylglucosaminidases from infant gut-associated Bifidobacteria release complex N-glycans from human milk glycoproteins.
      ), the ability to release N-glycans from host proteins has been mainly associated with endo-β-N-acetylglucosaminidase activity. In these bacteria, it seems that deglycosylation is initiated by the cleavage of the N-glycan between the two GlcNAc residues common to all N-glycans. Because the two first enzymes acting on the N2M3FX substrate (i.e. NixK and NixI) in Xcc were detected in the supernatant-enriched fraction of bacterial cultures, we can propose that degradation of the N2M3FX N-glycan is initiated outside the cell (Fig. 7). Possibly after cleavage of the peptide by the asparaginase AspG (XCC2914, proposed in the KEGG pathway database to cleave the N-glycan from the asparagine residue of N-glycoproteins), uptake of partially degraded N-glycans through the outer membrane could be achieved by Nix TonB-dependent transporters. Indeed, we have recently observed that these active transporters belonging to the GlcNAc utilization system are involved in the uptake of GlcNAc-containing complex molecules (
      • Boulanger A.
      • Zischek C.
      • Lautier M.
      • Jamet S.
      • Rival P.
      • Carrère S.
      • Arlat M.
      • Lauber E.
      The plant pathogen Xanthomonas campestris pv. campestris exploits N-acetylglucosamine during infection.
      ). This is in agreement with the proposed role of the TonB-dependent transporter GpdC of C. canimorsus in the uptake of the N-glycan moiety after cleavage by the endo-β-N-acetylglucosaminidase GpdG (
      • Renzi F.
      • Manfredi P.
      • Mally M.
      • Moes S.
      • Jenö P.
      • Cornelis G.R.
      The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG.
      ). Cleavage could continue in the periplasm because all Nix enzymes except NixG were detected in this cell compartment. The resulting chitobiose could be imported inside the cell through the MFS transporter NagP (
      • Boulanger A.
      • Déjean G.
      • Lautier M.
      • Glories M.
      • Zischek C.
      • Arlat M.
      • Lauber E.
      Identification and regulation of the N-acetylglucosamine utilization pathway of the plant pathogenic bacterium Xanthomonas campestris pv. campestris.
      ), whereas uptake of xylose through the inner membrane would be achieved via XylE (
      • Déjean G.
      • Blanvillain-Baufumé S.
      • Boulanger A.
      • Darrasse A.
      • Dugé de Bernonville T.
      • Girard A.L.
      • Carrére S.
      • Jamet S.
      • Zischek C.
      • Lautier M.
      • Solé M.
      • Büttner D.
      • Jacques M.A.
      • Lauber E.
      • Arlat M.
      The xylan utilization system of the plant pathogen Xanthomonas campestris pv campestris controls epiphytic life and reveals common features with oligotrophic bacteria and animal gut symbionts.
      ). NixG would further degrade chitobiose into GlcNAc, which would subsequently directly or indirectly inhibit repressors NagR and NagQ, leading to the induction of the expression of nag and nix genes (
      • Boulanger A.
      • Zischek C.
      • Lautier M.
      • Jamet S.
      • Rival P.
      • Carrère S.
      • Arlat M.
      • Lauber E.
      The plant pathogen Xanthomonas campestris pv. campestris exploits N-acetylglucosamine during infection.
      ,
      • Boulanger A.
      • Déjean G.
      • Lautier M.
      • Glories M.
      • Zischek C.
      • Arlat M.
      • Lauber E.
      Identification and regulation of the N-acetylglucosamine utilization pathway of the plant pathogenic bacterium Xanthomonas campestris pv. campestris.
      ).
      Figure thumbnail gr7
      FIGURE 7.Model of the N2M3FX N-glycopeptide degradation and utilization in Xcc ATCC33913 strain. NixK and NixI initiate deglycosylation of the N-glycopeptide outside the cell. We propose that AspG (XCC2914) would cleave the peptide moiety, and the resulting N2M2F N-glycan would be transported through Nix and/or other TonB-dependent transporters to the periplasm where NixJ, NixH, and NixE continue its degradation. Uptake of chitobiose (N2) through the inner membrane occurs via NagP, and NixG would be responsible for its degradation inside the cell. Free GlcNAc would then directly or indirectly inhibit repressors NagR and NagQ, leading to the induction of the expression of all nag and nix genes. Uptake of xylose through the inner membrane is performed by XylE. Transporters responsible for the uptake of xylose and mannose through the outer membrane and fucose and mannose through the inner membrane are not known. Free monosaccharides (fucose, mannose, GlcNAc, and xylose) would then be utilized for the metabolism of the bacterium. Numbers in circles indicate the order of action of Nix enzymes as determined in this paper. The boxes on the right indicate NixX-8His enzymes detected in total cell lysates (IN+PP), in periplasmic-enriched fraction (PP) and in the supernatant (OUT) of Xcc. F, fucose; M, mannose; N, GlcNAc; X, xylose; Nix, N-acetylglucosamine induced in Xanthomonas; TBDT, TonB-dependent transporter.
      In Xcc, the genes involved in N-glycan degradation are clustered and transcribed polycistronically (
      • Boulanger A.
      • Zischek C.
      • Lautier M.
      • Jamet S.
      • Rival P.
      • Carrère S.
      • Arlat M.
      • Lauber E.
      The plant pathogen Xanthomonas campestris pv. campestris exploits N-acetylglucosamine during infection.
      ), suggesting that they may act synergistically. A similar genetic organization has been recently described in Bacteroides species (
      • Nihira T.
      • Suzuki E.
      • Kitaoka M.
      • Nishimoto M.
      • Ohtsubo K.
      • Nakai H.
      Discovery of β-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase involved in the metabolism of N-glycans.
      ,
      • Nakayama-Imaohji H.
      • Ichimura M.
      • Iwasa T.
      • Okada N.
      • Ohnishi Y.
      • Kuwahara T.
      Characterization of a gene cluster for sialoglycoconjugate utilization in Bacteroides fragilis.
      ) and the corresponding cluster, important for growth on mucins in B. fragilis (
      • Nakayama-Imaohji H.
      • Ichimura M.
      • Iwasa T.
      • Okada N.
      • Ohnishi Y.
      • Kuwahara T.
      Characterization of a gene cluster for sialoglycoconjugate utilization in Bacteroides fragilis.
      ), has been proposed to be involved in sialoglycoconjugate utilization (sgu locus). However, some enzymes encoded by the sgu locus are different from those encoded by the Xcc N-glycan cluster. Indeed, the sgu locus codes for a sialidase (neuraminidase; GH33) and two other enzymes involved in sialic acid metabolism, whereas the Xcc N-glycan cluster codes for a β-xylosidase (GH3). These differences reflect structural specificities of mammal N-glycans as compared with plant N-glycans, respectively, and therefore suggest functional convergence to adapt these clusters to the ecological niches encountered by both bacteria.
      It has previously been described that glycans of N-glycoproteins can serve as nutrients for human pathogenic bacteria aiding in survival or persistence in vivo (
      • Roberts G.
      • Tarelli E.
      • Homer K.A.
      • Philpott-Howard J.
      • Beighton D.
      Production of an endo-β-N-acetylglucosaminidase activity mediates growth of Enterococcus faecalis on a high-mannose-type glycoprotein.
      ,
      • Byers H.L.
      • Tarelli E.
      • Homer K.A.
      • Beighton D.
      Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human α1-acid glycoprotein mediates growth of Streptococcus oralis.
      ,
      • Mally M.
      • Shin H.
      • Paroz C.
      • Landmann R.
      • Cornelis G.R.
      Capnocytophaga canimorsus: a human pathogen feeding at the surface of epithelial cells and phagocytes.
      ,
      • Renzi F.
      • Manfredi P.
      • Mally M.
      • Moes S.
      • Jenö P.
      • Cornelis G.R.
      The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG.
      ,
      • Collin M.
      • Svensson M.D.
      • Sjöholm A.G.
      • Jensenius J.C.
      • Sjöbring U.
      • Olsén A.
      EndoS and SpeB from Streptococcus pyogenes inhibit immunoglobulin-mediated opsonophagocytosis.
      ,
      • Burnaugh A.M.
      • Frantz L.J.
      • King S.J.
      Growth of Streptococcus pneumoniae on human glycoconjugates is dependent upon the sequential activity of bacterial exoglycosidases.
      ,
      • Garbe J.
      • Sjögren J.
      • Cosgrave E.F.
      • Struwe W.B.
      • Bober M.
      • Olin A.I.
      • Rudd P.M.
      • Collin M.
      EndoE from Enterococcus faecalis hydrolyzes the glycans of the biofilm inhibiting protein lactoferrin and mediates growth.
      ). We have recently observed that in Xcc, the N-glycan cluster participates in the degradation of GlcNAc-containing molecules of plant origin during infection (
      • Boulanger A.
      • Zischek C.
      • Lautier M.
      • Jamet S.
      • Rival P.
      • Carrère S.
      • Arlat M.
      • Lauber E.
      The plant pathogen Xanthomonas campestris pv. campestris exploits N-acetylglucosamine during infection.
      ), suggesting that deglycosylation probably occurs in planta during infection. Although N-glycans are widely distributed, these compounds represent only a small fraction of carbohydrates found in plants. This could explain the absence of phenotype in planta of the mutant deleted from the entire N-glycan cluster when inoculated into plants by piercing of the central vein (
      • Boulanger A.
      • Zischek C.
      • Lautier M.
      • Jamet S.
      • Rival P.
      • Carrère S.
      • Arlat M.
      • Lauber E.
      The plant pathogen Xanthomonas campestris pv. campestris exploits N-acetylglucosamine during infection.
      ). Although the oligosaccharide moiety could be the nutrient source, N-glycan degradative enzymes could also help bacterial proteases gain access to the peptide portion of the glycoprotein, resulting in an optimized nutrient supply. Indeed, glycoproteins become more sensitive to proteases after deglycosylation of N-linked oligosaccharides (
      • Berger S.
      • Menudier A.
      • Julien R.
      • Karamanos Y.
      Do de-N-glycosylation enzymes have an important role in plant cells?.
      ). In addition to their function in nutrition, the degradation of plant cell wall glycoproteins may lead to weakening of the cell wall, and better accessibility of cell wall polymers to degradative enzymes. Finally, this cluster could have an impact in signaling by activating or inactivating the function of proteins from the host. Indeed, in animals, many of the proteins involved in adaptive and innate immunity are glycosylated. Interestingly, the S. pyogenes, E. faecalis, and C. canimorsus human pathogens were shown to cleave the conserved N-glycan from human IgG (
      • Renzi F.
      • Manfredi P.
      • Mally M.
      • Moes S.
      • Jenö P.
      • Cornelis G.R.
      The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG.
      ,
      • Collin M.
      • Olsén A.
      Effect of SpeB and EndoS from Streptococcus pyogenes on human immunoglobulins.
      ,
      • Collin M.
      • Fischetti V.A.
      A novel secreted endoglycosidase from Enterococcus faecalis with activity on human immunoglobulin G and ribonuclease B.
      ), which, in addition to the potential role in nutrition, might impact immunity. In S. pyogenes, the EndoS protein that cleaves the chitobiose core of N-glycans (
      • Collin M.
      • Olsén A.
      EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG.
      ) interferes with the adaptive immune response and promotes bacterial growth in human blood (
      • Collin M.
      • Svensson M.D.
      • Sjöholm A.G.
      • Jensenius J.C.
      • Sjöbring U.
      • Olsén A.
      EndoS and SpeB from Streptococcus pyogenes inhibit immunoglobulin-mediated opsonophagocytosis.
      ). Similarly, plant pattern recognition receptors involved in innate immunity have N-glycosylations that are important for their function in pathogen-associated molecular pattern perception (
      • Häweker H.
      • Rips S.
      • Koiwa H.
      • Salomon S.
      • Saijo Y.
      • Chinchilla D.
      • Robatzek S.
      • von Schaewen A.
      Pattern recognition receptors require N-glycosylation to mediate plant immunity.
      ). Further investigations are now being undertaken to decipher on the role of the N-glycan cluster in Xcc during its life cycle, i.e. epiphytic life, entry into the leaf through hydathodes, infection, and survival on plant debris.

      Acknowledgments

      We are grateful to Patrice Lerouge (Université de Rouen) for stimulating discussion and for welcoming S. D. in his laboratory. We also thank François Lemauff and Coralie Boyer for technical assistance. We gratefully acknowledge Deborah Hinton and Nicolas Denancé for critical reading of the manuscript.

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